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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生命科學系所 > 依資料類型分類 > 碩博士論文 >  (一)綠藻 (Chlorella sorokiniana T-89) glutaredoxin的晶體結構分析 (二)Picrophilus torridus海藻糖合成酶的晶體結構解析與活性分析

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152375

標題: (一)綠藻 (Chlorella sorokiniana T-89) glutaredoxin的晶體結構分析 (二)Picrophilus torridus海藻糖合成酶的晶體結構解析與活性分析
(一)Crystal structure determination of glutaredoxin from Chlorella sorokiniana T-89 (二)Crystal structure determination and kinetics analysis of trehalose synthase from Picrophilus torridus
作者: 粘值菀
Nien, CHih-Yuan
Contributors: 劉俊宏
生命科學系所
關鍵字: 晶體結構分析;谷氧還原蛋白;海藻糖合成酶
Chlorella;glutaredoxin;trehalose synthase
日期: 2012
Issue Date: 2013-11-07 13:23:48 (UTC+8)
Publisher: 生命科學系所
摘要: (一) 細胞內的還原狀態對於細胞存活和增生等生理功能來說很重要,為了維持細胞內還原環境,細胞利用兩大系統thioredoxin (Trx) 和glutaredoxin (Grx) 作為氧化還原的調控。Grx屬於廣泛存在各種生物體內的小型抗氧化酵素,主要功能是透過本身cysteine殘基,以glutathione (GSH) 當作電子的提供者,還原兩種蛋白質中雙硫鍵型態,分別為因為氧化壓力所形成的蛋白質內雙硫鍵,以及藉由GSH與蛋白質形成的共價雙硫鍵。為了能更了解綠藻中Grx的氧化還原機制,我們解析氧化態與還原態Grx晶體結構,發現Chlorella sorokiniana T-89 的Grx 屬於thioredoxin fold superfamily,序列則類似人類的Grx2,並且觀察到氧化態Grx由活性區域Cys25和Cys28形成分子內雙硫鍵,而還原態Grx則有多個胺基酸和GSH之間形成交互作用力,這有可能是Grx在一般的情況下,GSH會嵌入Grx的催化活性位,保護催化活性位不受到自由基攻擊;另一種是推測,我們得到的還原態結晶是Grx以glutathionylation的狀態共價結合GSH,因為加入DTT,打斷了GSH與 Grx的共價鍵。我們根據NCBI BLAST和Dali server做進一步相似序列及結構的搜尋,選出人類、酵母菌以及大腸桿菌的Grx做比較,試著了解各個物種間結構與酵素和受質親和力的關係,再藉由比較氧化態與還原態Grx Grx-binding pocket,觀察在不同狀態下,胺基酸側鏈的方向移動與角度的改變,這項研究有助了解Chlorella sorokiniana T89 Grx在氧化還原平衡中,來達到抗氧化的作用。

(二) 海藻糖(trehalose)可以添加於食品、醫藥等產品中,在商業上具有重要的經濟價值。海藻糖合成酶(trehalose synthase)具有一步將受質麥芽糖轉化為海藻糖的酵素活性,因此海藻糖的生產因為主要以海藻糖合成酶為主。到目前為止有許多微生物物種的海藻糖合成酶的酵素活性相關研究,但是還沒有海藻糖合成酶的晶體結構被發表,因此本實驗嘗試解析出一株嗜熱嗜酸性的古生菌的海藻糖合成酶(Picrophilus torridus trehalose synthase, PTTS)晶體結構,並做結構分析,現在仍在繼續進行晶體結構各個數據的最佳化,以便得到較具可信度的蛋白質結構。另一方面也藉由分析PTTS酵素動力學性質,以便於更加了解酵素的表現與活性。
(一)Intracellular redox status plays an important role in cell functions, such as cell survival and proliferation. To maintain a reduced intracellular redox state, cell utilizes protein redox systems, thioredoxin (Trx) and glutaredoxin (Grx) systems. Glutaredoxins are small dithiol proteins, and can be found in many prokaryotes and eukaryotes, including humans. This system catalyzes thiol-disulfide exchange reactions or protein-mixed glutathione disulfides reduction to maintain thiol redox status of the cytosol by using glutathione (GSH) as the electron donor. To gain more knowledge on redox regulation in the single-celled green algae Chlorella, the recombinant Grx from Chlorella sorokiniana T-89 was crystallized in both oxidized and reduced states. The overall structure of Grx resembles the fold of the Trx superfamily, and Grx shares sequence similarity with human Grx2. In the active site of the oxidized Grx, Cys25 and Cys28 forms a disulfide bond. In the reduced Grx structure, glutathione makes several polar contacts with Grx. In normal state, GSH embedded in Grx catalytic active site, it may protect the catalytic activity site from free radical attacked. In the reduced status Grx would alter some side-chain conformation to accommodate GSH. We also used NCBI BLAST and the Dali the server to do a further search for similar sequences and structures of different species of Grx. We try to understand the relationship of structure and enzyme and substrate affinity between the various species This study would be very helpful for understand how Chlorella sorokiniana T89 Grx to regulate a reduced intracellular redox state to defense of oxidative stress.

(二)Trehalose (trehalose) is an important economic value in the business, because it can be added to food, medicine and other products. Trehalose synthase can catalyze the conversion of maltose into trehalose in one step. Therefore, trehalose synthase is the main method to product trehalose. So far, trehalose synthase activity from many microbial species was published, but the crystal structure of the trehalose synthase was not published yet. So this experiment is to try to determine the protein structure of trehalose synthase from a thermophilic and acidophilic archaea Picrophilus torridus, and structural analysis. Now We are trying to optimizethe the crystal structure data. The other hand, the analysis of the PTTS enzyme kinetic characteristics can understand the activity of the enzyme.
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